Designing of multi-epitope chimeric vaccine using immunoinformatic platform by targeting oncogenic strain HPV 16 and 18 against cervical cancer

Cervical cancer is the most common gynaecological cancer and reaches an alarming stage. HPVs are considered the main causative agents for cervical cancer and other sexually transmitted infections across the globe. Currently, three prophylactic vaccines are available against HPV infections with no therapeutic values. Due to a lack of effective therapeutic and prophylactic measures, the HPV infection is spreading in an uncontrolled manner. Next-generation of vaccine is needed to have both prophylactic and therapeutic values against HPV. Here first time we have designed a multi-epitope chimeric vaccine using the most oncogenic strain HPV 16 and HPV 18 through an immunoinformatic approach. In this study, we have used the L1, E5, E6 and E7 oncoproteins from both HPV 16 and HPV 18 strains for epitope prediction. Our recombinant chimeric vaccine construct consists, selected helper and cytotoxic T cell epitopes. Our computational analysis suggests that this chimeric construct is highly stable, non-toxic and also capable of inducing both cell-mediated and humoral immune responses. Furthermore, in silico cloning of the multi-epitope chimeric vaccine construct was done and the stabilization of the vaccine construct is validated with molecular dynamics simulation studies. Finally, our results indicated that our construct could be used for an effective prophylactic and therapeutic vaccine against HPV.

www.nature.com/scientificreports/ additional impact on LR-HPV 6 & HPV 11 and showed efficacy in preventing anogenital warts caused by HPV 6 & 11 (https:// www. who. int/ immun izati on/ hpv/ vacci nes/ en/). However, none of them have therapeutic effects 9,10 . Due to the high prevalence and mortality with HPV associated cervical cancer, an effective therapeutic HPV vaccine or a vaccine having both prophylactic and therapeutic properties is urgently required for the clearance of the virus from the host. Peptide-based vaccines have certain advantages as they are easy to produce and transport, have high selectivity, multivalency competence and have accessibility to the epitope. With the advancement in genome sequencing techniques, potential B and T cell epitopes can be predicted and have a promising outlook for the development of the peptide-based vaccine against any infectious agent or disease and/or cancers. At present, a branch of bioinformatics, immunoinformatics, used for the identification of effective T-& B-cell immunogenic epitopes from the antigenic peptide by different software which aids us to classify cost and labor effective immunogenic peptides by eluding the non-immunogenic sequence. These methods aid as low-cost by saving the cost of synthetic peptide expense of and working time 11,12 .
The polymorphism in host genetics can impact the immune response to a pathogen in the target population. In view of the polymorphic nature of major histocompatibility complex (MHC) class I and II alleles they bind towards specific repertoire, short peptides (epitopes), from the processed pathogen and presentation of these complexes to T cells is very important for the development of effective cytotoxic T lymphocytes (CTL) response against the pathogen 13 . The present study focused on the development of the effective chimeric vaccine having both prophylactic and therapeutic effects against the two HR-HPV (HPV 16 & 18) by reverse vaccinology approach. Here we have analyzed the L1, E5, E6 and E7 proteins of both HPV 16 and 18 by employing various algorithms for the prediction of the best T-cell and B-cell epitopes. Next, we have constructed a multi-epitope chimeric construct and evaluated all physiochemical parameters along with its antigenicity and allergenicity. Furthermore, we have done in silico cloning and checked the stability of our construct through MD simulation. The complete design and methods were used in this study are depicted in Fig. 1.

Results
T lymphocyte epitopes. Since epitopes on T cells are bound to MHCs, hence their interaction can be modeled accurately on the basis of certain algorithms. Both MHC-I & MHC-II epitopes are needed to make an immunogenic vaccine construct. Here, we have predicted the potent MHC-I & MHC-II epitopes using IEDB server on the default parameters.

MHC-I epitopes prediction.
For MHC-I, the prediction of the epitopes of L1, E5, E6 & E7 proteins of HPV 16 & 18 has been done for the 27 reference alleles. The epitopes having a percentile score less than 0.5 have been short listed. The epitopes having the antigenicity score greater than 0.4 were considered as probable antigens. HPV 16. A total of 155, 12, 22 and 17 epitopes having the percentile score < 0.5 in L1, E5, E6 & E7 proteins of HPV 16, respectively, has been selected for further analysis. These predicted epitopes were further analyzed for the antigenicity score and MHC-I immunogenicity. On analysis, 18, 03, 05 & 04 were found to have antigenicity score 0.4 and predicted as probable antigen for L1, E5, E6, & E7 protein, respectively (Table 1).    (Tables 5 and 6). The final construct was subjected to ABCpred for the prediction of the potential B cell epitope at a cut-off of 0.8 and 24 potential epitopes were predicted in the multi-epitope www.nature.com/scientificreports/   Allergenicity and antigenicity. The allergenic property of the multi-epitope chimeric vaccine construct was predicted by AllerTOP v2.0 and found to be non-allergenic behavior. The predicted antigenicity score of this construct was 0.5883 by using Vaxijen v2.0 at a threshold value of > 0.4%, which shows the antigenic nature of the construct.

Multi-epitope chimeric vaccine construction and its characterization.
The multi-epitope chimeric vaccine construct favored secondary and tertiary structure. The 411 amino acid long multi-epitope chimeric vaccines construct was analyzed for secondary structure, out of 411 amino acids, 200 (48.66%) formed alpha helix, 69 (16.78%) β-strands and coil were formed by 142 (34.54%) (Fig. 3a). I-TASSER uses 10 threading templates which showed good Z-score values (ranging from 0.62 to 3.54) and predicted 5 models. The model was selected on the basis of a high C score (higher value indicates higher confidence) which typically ranges from − 5 to 2 (Fig. 3b). The selected model was analyzed for the Ramachandran plot using the SAVES server and found 93.8% of residues in the most favored regions and additional allowed regions (Fig. 3c).

Toll-like receptor (TLR)-3 and TLR4 established the interaction with multi-epitope chimeric vaccine construct.
The multi-epitope chimeric vaccine construct with the selected epitopes was docked with TLR3 (2A0Z) and TLR4 (3FXI) and showed good interaction. The docked structure along with molecular interactions between docking complex of vaccine construct and TLR3 & TLR4 were visualized in the LigPlot (Fig. 4a,b).

MD simulation studies show a stable interaction between ligand and its receptor.
The molecular dynamics analysis was performed to access the binding interactions and flexibility of the binding site. MD simulations were done for 100 ns on each complex (total 200 ns) and the stability of the simulation was evaluated using root mean square deviation (RMSD). The RMSD values reveal the structural changes that occurred during the MD. The RMSD plots for all proteins indicated that each system got stabilized quickly and then remained stable throughout the simulation time as evidenced by the movement of the RMSD curve within the 2 Å (Fig. 5a,b). These plots suggested that each system was quite stable for further study. www.nature.com/scientificreports/ The root-mean-square fluctuation (RMSF) study was done to get an insight into the flexibility of individual amino acids during the simulation. The amino acid residues taking part in important interactions are generally constrained and show less flexibility as compared to other amino acid residues in the complex. It can be seen from RMSF plots (Fig. 5c,d) that the beta-sheets of TLR3 and TLR4 are showing less fluctuations (wavy pattern) due to strong hydrogen bonding between them. The other part of HPV is showing greater fluctuations. A comparison of the two plots indicates that the complex with TLR3 is showing comparatively lesser fluctuations indicating better binding among the interaction partners and a stronger complex.
The chimeric vaccine construct-TLR3 salt-bridge analysis revealed residues at the binding interface show strong interactions especially ARG348_chimeric vaccine construct-GLU533_TLR3, ASP246_chimeric vaccine construct-LYS41_TLR3, ARG380_chimeric vaccine construct-GLU306_TLR3 were found to be quite stable with percent occupancy of 72, 63 and 55 respectively. It can be seen in Fig. 5e that these salt bridges anchor the two proteins at three distinct points. In case of HPV-TLR4 complex, the binding interactions were less strong as compared with the chimeric vaccine construct-TLR3 complex. The ARG598_TLR4-GLU330_chimeric vaccine construct, ASP453_TLR4-LYS26_chimeric vaccine construct, GLU154_TLR4-ARG36_chimeric vaccine construct interactions showed however some stabilizing interactions with > 40% occupancy. The salt-bridges are shown in Fig. 5f. The results clearly showed the complex HPV-TLR3 is much more stable than chimeric vaccine construct-TLR4.
In silico cloning of the construct in pcDNA3.1/V5/His-Topo vector. The vaccine candidate was processed for the codon optimization for the maximal level of protein expression in E. coli using Jcat server and the optimized vaccine candidate had 1233 nucleotide and the GC content of 67.8%, which showed the high expression of the vaccine candidate. The plasmid vector was prepared using the SnapGene software into pcDNA3.1/ V5/His-Topo (Fig. 6).
Immune simulation analysis shows an effective cytokine and antibody response. The result of the C-ImmSim studies showed the effective immune responses of the vaccine candidate which showed the high level of the IgM and also showed the increased level of the immunoglobulin (IgG1 + IgG2, IgM and IgG + IgM) expression and T helper cell and cytotoxic T cells. The production of the cytokines & interleukins was identified along with increased B-& T-cells (Fig. 7).

Discussion
Cervical cancer caused by HPV has reached to its alarming stage and become a public threat globally 14 . The current management of this disease is not satisfactory and mainly depends on three prophylactic vaccines 15 . So far, no therapeutic vaccine is available against HPV. Hence, a vaccine having both prophylactic and therapeutic properties will help not only to prevent the HPV infection but should be used to treat the established infection.
Nowadays, immunoinformatics, a branch of bioinformatics, offers new tools for the identification and design of epitopes against specific antigens, which could be used as an ideal target for the development of vaccine against specific viral or pathogenic infections 16,17 . Immunoinformatics, helps in the identification of effective epitopes which can activate both cell-mediated or humoral immunity against specific viral or pathogenic infections. In 2003, Adu-Bobie et al., developed the first vaccine against Neisseria meningitides by using immunoinformatics approach 18 . Many research articles showed the importance of the highly immunogenic epitopes for CD8 + and CD4 + CTLs is required in a vaccine for persuading a strong immune response 19,20 . The peptide (generally 8-11 residues) presented by the MHC-I molecules intracellularly recognized by CD8 + CTLs, whereas the MHC-II recognized extracellular-originated peptides (generally 10-30 residues) by CD4 + Helper T lymphocytes (HTLs). The ideal peptide length for MHC-I and MHC-II is 9 residue & 12-16 residue, respectively. The strong immune response generated by a vaccine depends on the interaction of peptide-MHC complex (pMHC) and T cell receptor 19,21 . Also, This reverse vaccinology approach has various advantages due to its less time-consuming property, cost-effectiveness, more accuracy and safety. Therefore, most of the research has been done taking this www.nature.com/scientificreports/ approach in different organisms such as viruses, bacteria, and parasites, for the identification and development of multi-epitope vaccines [22][23][24][25][26][27][28][29] .
In the present study, the epitopes for the MHC-I & MHC-II were predicted for the L1, E5, E6 & E7 proteins of two highly oncogenic strains of HPV i.e. HPV 16 & HPV 18. These selected proteins of HPV 16 & HPV 18 play an important role in the viral structure, cell transformation and immune evasion to induce malignancy in the cells [30][31][32] . The chimeric vaccine construct prepared in the study has also shown 16 potential B cell epitopes when submitted to ABCpred server. The most potent epitopes were LQFIFAAYKFGFPDTS, PGSIFYHAGSSRLLTV, & PGTAFTVYVFCFLLPM having score more than 0.90, 0.89 & 0.88, respectively. Further we have made multiepitope chimeric construct with the help of adjuvants and linkers with appropriate position.
The final vaccine construct has the molecular weight of 46.24 kDa with chemical formula C 2177 H 3223 N 529 O 564 S 13 and has a theoretical isoelectronic point (pI) was 8.67, which suggested the basic nature of the prepared vaccine construct. The aliphatic index and grand average of hydropathicity (GRAVY) of the construct were 91.07 and 0.186, respectively, which suggested that protein is thermostable & hydrophilic in nature. Sympathetic    Earlier, several attempts have been made for the development and identification of multi-epitope vaccines against HPV such as the use of virus-like particles (VLPs) based on L1 protein 33 and L2-based vaccination 34,35 . In another study by Negahdaripour et al., predicted epitopes from the HPV 16 L2 protein 36,37 while other studies used L1 protein of HPV 16 for epitope prediction 38 . Overall, most of the studies were performed using E6 and E7 protein as key molecules for epitope prediction due to various reasons.
The search for a better vaccine candidate is still under consideration. Earlier, most of the studies were done by targeting E6 and E7 proteins of HR-HPV 16 and 18. However, none of them reached clinical trials. The majority of the previous studies have focused on the E6, E7 oncoproteins and L1 capsid protein of HPV. However, apart from the oncogenic E6, E7 protein and highly immunogenic L1 protein, E5 is crucial in HPV pathogenesis. E5, along with E6 and E7 is responsible for the transformation of normal cells, downregulation of MHC I expression, protein trafficking through ER and preventing the acidification of endosomes [39][40][41][42][43] Recently, Namvar et al. predicted epitopes from the E5 and E7 proteins of HPV 16/18/31/45 44 implying the future potential of targeting HPV E5 protein.
Here, for the first time we have analyzed and taken the major oncoproteins (E6 and E7), highly immunogenic L1 capsid protein and also, E5 protein from both the oncogenic strains of HPV (HPV16 &18) and prepared    www.nature.com/scientificreports/ a multi-epitope chimeric vaccine through in silico approach. Our multi-epitope construct has peptides like CYSLYGTTL, ISEYRHYCY, KLPDLCTEL, LLMGTLGIV and TLQDIVLHL that have been experimentally validated [45][46][47][48][49] . In addition, the first HPV 16 E7 predicted epitope in our study RAHYNIVTF, was also recognized by Feltkamp et al. 1993 who were the pioneers of HPV epitope studies 50 . Taken together, our results showed the chimeric construct has the ability to induce both cell-mediated and humoral response and consisting all immunogenic, physicochemical and structural properties which are required for ideal vaccine design. The immune stimulation analysis suggested the efficiency of our construct to initiate an effective cytokine response and B cell response. Finally, our multi-epitope chimeric construct can be used in both prophylactic and therapeutic purposes in in-vitro/in-vivo studies against HPV infection. In addition, further validation will be required for the use of our construct in combination with other cancer immunotherapy approaches to target HPV-induced malignancies.

Material and method
Retrieving of protein sequences. The  Prediction of MHC Class I immunogenicity. The identified MHC class I epitopes were further examined and confirmed for the immunogenicity by using another tool i.e. IEDB MHC Class I immunogenicity (http:// tools. iedb. org/ immun ogeni city/). This tool only analyzed and validated on 9 mer peptides.

Prediction of antigenicity & IFN-γ inducing epitope.
Vaxijen, an online tool was used to check the antigenicity in predicted epitopes 51 . Further, to confirm whether selected epitopes have the ability to induce IFN-γ, we have used IFN epitope server (https:// crdd. osdd. net/ ragha va/ ifnep itope/ predi ct. php) in this study 52 . In our analysis, we have performed Motif and SVM hybrid algorithms and IFN-γ versus non IFN-γ model for prediction 52 . Preparation and Physiochemical properties of the vaccine construct. A chimeric vaccine construct of HPV 16 and HPV 18 having MHC class I & II predicted immunogenic and conserved epitopes were analyzed together with linkers to get the final vaccine construct using β-defensin adjuvant. It has been well established the role and properties of β-defensin as an adjuvant against viral infections 24,53 , which was associated with EAAAK linker at N terminal. The AAY linker was associated with MHC-I epitopes while MHC-II epitopes were linked with GPGPG linkers. The Protparam server was used to compute and validate the physiochemical properties of the final vaccine construct.
Prediction of B-cell epitopes. In the reverse vaccinology approach, a successful vaccine must also induce a strong B-cell mediated humoral immune response. Hence, it is of utmost to show that chimeric vaccine construct is able to induce protective humoral immunity. Here, for B cell epitope prediction the artificial neural network-based ABCpred server (http:// www. imtech. res. in/ ragha va/ abcpr ed/ ABCsu bmiss ion. html) was employed. All the parameters were setup in default conditions but selected the epitope having score more than 0.8 54 .
Prediction of Allergenicity. Proteins and peptides are well known to induce allergenic reaction. We have used an online tool AllerTOP v2.0 (https:// www. ddg-pharm fac. net/ Aller TOP/) to check the allergenicity of the chimeric vaccine construct.
Prediction of secondary and tertiary structure. The Psipred, which is an online server was used to predict the secondary structure of prepared chimeric vaccine construct which mainly uses primary amino acid sequences in specific manner (http:// bioinf. cs. ucl. ac. uk/ psipr ed/). For the prediction of the tertiary structure of the chimeric vaccine construct, the online freely available server I-TASSER server (https:// zhang lab. ccmb. med. umich. edu/I-TASSER/) was used which utilizes sequence-to-structure-to-function paradigm. The last five community-wide CASP experiments ranked I-TASSER server as the top server for the prediction of protein structure 24,55 . Validation of the tertiary structure. PDBsum, a pictorial database was used to validate the tertiary structure which is based on to generate Ramachandran plot (http:// www. ebi. ac. uk/ thorn ton-srv/ datab ases/ pdbsum/ Gener ate. html) that gives an at-a-glance impression of each 3D structure contents submitted in the PDB. Finally, the validation of the 3D structure is vital for the protein model, as it can detect potential errors in modeled 3D structure. MD simulation. In the current study, the preliminary topology and coordinates for the complexes were generated in VMD version 1.93 57 . The complexes were prepared and solvated in a rectangular water box (TIP3P) with a buffering distance of 10 Å. Ions were added to ensure the electro-neutrality of the solvated system. SETLE algorithm was used for the water molecules model system. The associated system topology and coordinates were generated by applying charmm34 force field parameters for MD simulation. The MD simulations were executed using NAMD version1.9 58 . Prior to the simulation, the system was properly minimized with a stepwise minimization protocol. Firstly, the water molecules and ions were minimized, followed by hydrogen atoms and the side chains of the complex. The side chains were minimized for 100,000 steps while the backbone atoms and the bond lengths of hydrogen atoms were kept fixed. Thereafter, all the atoms were allowed to relax freely and the whole system was energy-minimized for 100,000 steps with nominal restraints on C-alpha atoms and DNA backbone atoms (10 kcal/mol) to prevent any abrupt change in structure. Subsequently, an equilibration protocol was followed where the system was heated gradually from 0-310 K in steps of 30 K with a canonical ensemble (NVT). At each step, a 20 picosecond (ps) simulation was run to allow the system to adjust to the temperature.
Once the system attained 310 K, an isobaric and isothermic ensemble (NPT) was applied for a period of 100 ps with a constant pressure of 1.0 bar using Langevin dynamics0. Finally, the applied restraints on C-alpha atoms and DNA were removed and the system was equilibrated for 1 ns at 310 K using Langevin piston coupling algorithm. During the whole simulation, the Particle Mesh Ewald sum algorithm (PME) algorithm was used to calculate the long-range electrostatic interactions, the hydrogens were constrained using SHAKE algorithm. After equilibration, a production run was done on each complex for 100 ns. The analyses of the MD trajectories were performed to get an insight on the structure and dynamic behavior of all complexes. The trajectories were analysed for root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonds, and salt bridges in VMD.
In silico cloning and optimization of designed vaccine candidate. Java Codon Adaptation Tool (JCat) server (http:// www. jcat. de/) was used for codon optimization and reverse translation to check translation and efficiency of cloning of the multi-epitope vaccine construct. The protein expression level was assessed by using codon adaptation index (CAI) and the percentage GC content generated in JCat output 59 . The optimized sequence of the vaccine was then cloned into pcDNA3.1/V5/His-TOPO/LacZ vector using DNASTAR (https:// www. dnast ar. com).
Immune simulation. In order to characterize the effective immune response generated by the prepared vaccine construct, an online simulation server, C-ImmSim (http:// 150. 146.2. 1/C-IMMSIM/ index .php), was used 61 . This server uses a position-specific scoring matrix (PSSM) to predict immune epitope and predict the immune interaction by using machine learning. It predicted both cellular and humoral immune in mammalian immune system 60,61 . The server was used on the default parameter with simulation volume 50 and the simulation steps 1000 with three injections of the predicted vaccine construct at an interval of 4 weeks.